importing transition only data to scan for PTM diagnostic ions

importing transition only data to scan for PTM diagnostic ions janelle hancock  2022-08-16 19:33

Dear Skyline developers,
Thanks for developing and continuing to update this great tool.

We would like to be able to identify scans in which particular diagnostic ions occur, regardless of the precursor. Our modifications are Lysine linked ubiquitin or Sumo type peptide remnants. The mods are 4-6 amino acids long. We are running an Q exactive HF in standard TopN DDA mode. The modification itself is a peptide, and upon HCD fragmentation produces a predictable pattern on neutral losses (which remain attached to the parent peptide) and released diagnostic b ions. I've attached an example spreadsheet of one such tryptic remnant, from tryptic digest of the human sumo-like protein UFM1.

The spectra for these modified peptides is normally quite complex and we don't get many good matches using PD or maxquant. If possible I would like to use skyline to identify what scans contain these diagnostic b-ions. Once I know that, I can make an inclusion list to re-run the same samples, acquire better quality spectra, enabling us to get and ID and modification site assignment.

Can you please tell me if I can use skyline to analyze transitions only, without considering what precursor they came from?


Nick Shulman responded:  2022-08-16 19:56
If you want to tell Skyline that every MS2 spectrum might contain your molecule of interest, regardless of what was isolated in that MS2 spectrum, you should go to:
Settings > Transition Settings > Full Scan
and set the "MS/MS filtering Acquisition method" to "DIA" and set the "Isolation Scheme" to "All Ions".

I imagine that the chromatograms that you get when you do that will not be useful. That is, they will not have any sort of consistency between spectra, so peak finding or area integration will not work very well.

Skyline is not a good tool for looking for things that appear in only one spectrum. Skyline is good at quantifying things that have well shaped chromatogram peaks. Skyline also does not do a good job with DDA because the same precursor does not get sampled at any sort of reliable intervals.

By the way, you might also find the "Special ions" list helpful at "Settings > Transition Settings > Filter". That is where you would tell Skyline about ions that you would like Skyline to look for regardless of what they were attached to. The usual case where people use this are TMT tags, but it also sounds like it might be similar to what you are looking for.

What you are asking for sounds a lot more like a peptide search engine with an open modification. Then, after you have a list of things that might be attached to your fragment ions, you can tell Skyline to extract chromatograms from these targets.

I might not have understood your question. Also, someone else on this support board might have better ideas about what you could do.
-- Nick
Juan C. Rojas E. responded:  2022-08-17 00:36
I'll jump in here because it is a perfect excuse to place a reminder about the open request for reporter ion support for annotating them in the MS/MS library viewer :)

With regards to the neutral losses consideration for search engines, Janelle, I am not sure what tools you have access to, but for PEAKS Studio there are ways of editing the program to consider custom neutral loss fragment ions that should help you in annotating fragment ions specific to your PTM. I think through FragPipe+MSFragger you can also provide custom fragment ions to consider, but I haven't had much success with this tool. Anyway, in both cases I advice you to reach out to the developers (the MSFragger team are very quick to respond and the PEAKS Studio as long as you have an active license).

In Skyline I know you can add custom neutral losses to the PTMs of interest which it will be able to annotate them in the spectral library viewer and compute them for fragment EICs. However, as Nick pointed out, this would only be useful if you have the IDs a priori, which from what you are describing is the main issue here.

Juan C.

PS: best of lucks with the PTM adventures!